IL-4 and IL-6 are pleiotropic cytokines that can act independently or synergistically to regulate lymphocyte growth and differentiation. Both of these factors are produced by cultures of Th2 cells but it is uncertain whether they are secreted simultaneously by a single cell. We used a sensitive and specific ELIspot assay to identify individual cells spontaneously producing IL-4 or IL-6 under physiological conditions in vivo. The fraction of cells producing these two lymphokines was regulated concurrently in cloned Th2 cells but varied independently in the lymphoid organs of normal BALB/c mice. A sandwich ELIspot assay was developed to determine whether individual cells simultaneously produced both cytokines. Thirteen per cent of cells in the spleen and 25% of those in the bone marrow of 10-week-old BALB/c mice secreting either IL-4 or IL-6 concurrently produced both of these lymphokines. These observations establish a physiological mechanism by which the concurrent production of synergistic cytokines is regulated in a given micro-environment.